Construction of a bacterial artificial chromosome library 
for pea (Pisum sativum L.)

Coyne, C.J.,                                                                                     USDA-ARS Plant Introduction

                                                                   Washington State University, Pullman, WA 99164-6402

Meksem K., Lightfoot, D.A.,                                          Dept. of Plant, Soil, and General Agriculture

                                                                  Southern Illinois University, Carbondale, IL 62901‑4415

Keller, K.E.,  Martin, R.R.,                                USDA-ARS Horticulture Crops Research Laboratory

                                                                                                                      Corvallis, OR 97330

McClendon, M.T.,                                               Dept. of Horticulture, Washington State University

                                                                                                              Pullman, WA 99164-6402

Inglis, D.A.,                                                                          Mount Vernon Research and Extension

                                                          Washington State University, Mount Vernon, WA 98273-9761

Storlie, E.W.  and                                                        Department of Biological & Physical Sciences

                                          Univ. of Southern Queensland, Toowoomba, Queensland 4350, Australia

McPhee, K.E.                                                    USDA-ARS Grain Legumes, Dept. of Crop Science

                                                                   Washington State University, Pullman, WA 99164-6402

Introduction

      One of the major constraints to fully utilizing the U.S. plant germplasm resources is the paucity of effective techniques to efficiently discover new alleles useful to crop breeding (12).  Several key genomic technologies relevant to germplasm collections, however, have been developed (1).  Information gathered using this functional genomics approach may be useful in identifying disease and stress resistance alleles in U.S. pea germplasm accessions as a molecular tool for efficient curation of the collection.  Because the USDA-ARS  National Plant Germplasm System (NPGS) encourages its system of repositories to embrace new technologies to maximize the conservation of genetic variation for economically important traits in each collected crop species, one such biotechnology tool, a bacterial artificial chromosome (BAC) library of the pea genome, has the potential to provide new opportunities for the development and management of pea genetic resources.

      Our objective was to construct a bacterial artificial chromosome (BAC) library of Pisum sativum from the germplasm line PI 269818 of the NPGS in the binary vector pCLD04541, (V41).  The binary vector potentially allows for the direct transformation of candidate pea gene BACs into plants to verify the phenotype (11).  Pea has a number of stable and single gene traits useful to breeders, including resistance to pea seedborne mosaic virus (PSbMV) conferred by sbm-1.  PSbMV has been disseminated worldwide in infected seed (7).   The NPGS places a high priority to maintain and distribute Pisum germplasm tested free of seed-borne pathogens such as PSbMV.  Thus, virus-free seed is critical to the mission of the cool season food legume program of the Western Regional Plant Introduction Station at Washington State University.  The library will be useful for (i) positional cloning of disease resistance genes, such as sbm-1 and other economic traits (2), (ii) targeted microsatellite development to economic traits of interest (4, 9), and (iii) use of the microsatellite markers for fingerprinting PI accessions to eliminate duplication of pea germplasm and assess genetic diversity in the collection (5).

Materials and methods

Plant materials

      Seed of PI 269818 was obtained from the National Plant Germplasm System.  For DNA isolation, plants were grown in a growth chamber in soiless mixture for 14 days, with 16 h light, 8 h dark at 22°C. Two weeks after germination, seedlings were grown in continuous dark for 3 days to reduce the carbohydrate content of the leaves.

Vectors and DNA preparation

      The BAC vector V41 (pCLD04541) was developed by Dr. J.D.G. Jones (8) and generously provided by Dr. H. Zhang (Texas A & M University, USA). The V41 is a binary vector for Agrobacterium-mediated plant transformation and has been shown to be capable of stable maintenance of large plant DNA (9, 11).  The method for high molecular weight (HMW) DNA preparation from plant nuclei was described by Zhang et al. (13). Nuclei were prepared from 25 g of leaves and embedded in 12 ml of 0.5% (w/v) low melting point agarose plugs.  The plugs  were cut into 12 pieces, partially digested for 10 min at 37°C with HindIII (Gibco-BRL, MD) and BamHI (Gibco-BRL, MD).  Restriction enzyme concentration was optimized for each set of HMW DNA isolations.  Test digests were performed in seven increment steps of 0.3 to 5 units of enzyme per 1 mg of DNA followed by separation using PFGE (pulsed field gel electrophoresis) on a CHEF DRIII  (Bio-Rad, Hercules, CA).  Each digestion was carried out in a 200 ml total volume. Reactions were stopped by adding 20 ml of ice cold 0.5 M EDTA pH 8, on ice.

Construction and storage of the BAC library

      The vector V41 was purified with Qiagen plasmid purification kit, followed by two cesium chloride gradients. The vector was digested (HindIII or BamHI ) and then dephosphorylated to prevent self ligation. Partially digested HMW DNA was size selected on 1% (w/v) pulsed field low melting point agarose gels in 0.5x TBE (45 mM Trizma base, 45 mM boric acid and 1 mM EDTA, pH 8.3) by PFGE. Two size-selections were performed to increase the average insert size of the BACs, and to eliminate small DNA fragments trapped in the HMW fraction. The first PFGE size selection was performed for 18 h at 11°C with constant pulse time of 90 s, at a 120° angle and 6 V/cm. The second size-selection was performed for 12 h at 11°C with an initial pulse time of 1 s, a final pulse time of 10 s, at a 120° angle and 6 V/cm.

      After the second PFGE, two methods were used to isolate the size‑selected DNA from the agarose gel: digestion of the agarose by b-agarase (Gelase, Epicentre, Madison, WI) or by membrane dialysis.   For ligation into vector V41, the molar ratio of the vector to the insert DNA was 4:1. After 24 h incubation at 11°C, ligated DNA was transformed by electroporation into the E.coli strain ElectroMAX DH10B using Gibco BRL Cell Porator and Voltage Booster system (Gibco BRL, Grand Island, NY). Recombinant transformants were selected on LB agar plate containing 15 mg/l tetracycline, 0.5 mM IPTG and 50 mg /ml X-gal. After 24 h incubation at 37°C, individual white colonies were picked into 384 well plates containing LB freezing medium using Flexsys robotic workstation (Genomic Solutions, Ann Arbor, MI) or by hand using sterile wooden toothpicks. The plates were incubated at 37°C for 14 h and stored at -80°C. The average insert size was assayed after plasmid DNA extraction from BACs using the method of Sambrook et al., (10) restriction digestion with NotI to free the DNA insert from the vector and size separation by PFGE.  The fragments were stained with ethidium bromide and visualized with ultraviolet light.

      Working copies of the BAC library were produced using a Flexsys robotic workstation and by hand using a 384 pin replicator (Nunc, Naperville, IL).  The replicator was used to spot 6912 clones of the library onto Hybond N⁺ filters (Amersham-Pharmacia, Piscataway, N.J.).  The inoculated filters were placed on plates containing LB agar and 25 mg tetracycline and incubated overnight at 37°C.  The filters were prepared for hybridization with random hexamer ³²P-labeled DNA probes of oligomer repeat primer (ATATATATATATATATATAT) (10).

Results

      The isolation of DNA using the method of Zhang (13) resulted in concentrated high molecular weight DNA.  Optimization of restriction enzyme concentration indicating the use of very low concentrations of enzyme (0.1-1 units per 1 mg of DNA) to achieve the desired partial digest of the DNA.  Both methods of the isolation of size-selected DNA fragments resulted in high numbers of fragments for ligating into V41.  The first method of b-agarase enzymatic digestion of the agarose was used for the first 20,000 packaged BAC clones and the method using membrane dialysis was used for the next 30,000 cloned fragments.  Partial DNA digests using HindIII produced high quality and quantity transformable ligations than did restriction digests and ligations than with BamHI digests (data not shown).  Therefore, we used only HindIII partial digests of the HMW DNA to complete the 50,000 BACs for the one genome equivalent coverage of pea.  We tested using one size selection PFGE of the HMW DNA before ligation.  The number of transformants was very high (20,000 per ml ligated DNA/ml of competent cells).  However, the average insert size (£60 kb) would increase the number of clones significantly to achieve good coverage of the pea genome.  We also ran the two size-selection PFGE conditions on one gel.  This also resulted in an undesirable decrease in average insert size.  Therefore, two size-selection gels were used to prepare all the DNA for ligation into V41.

      Using the parameters described, we packaged 50,000 large-insert BACs from the pea genome.  BAC insert DNA was isolated by miniprep, digested with the restriction enzyme NotI, electrophoresed using the PFGE, and visualized with ethidium bromide and ultraviolet light to estimate the average insert size (Fig. 1).  The distribution of seventy-nine BACs indicate an average size of 110 kb (Fig. 2).  The library has been duplicated to provide a working copy for preparation of filters for hybridization.  The oligomeric repeat primer hybridized to the BAC inserts indicating the microsatellite strategy may be useful in pea.

Discussion

A BAC library of Pisum sativum, representing one genome equivalent, has been constructed of partially HindIII-digested DNA from the pea germplasm line PI 269818 from the NPGS in the binary vector pCLD04541.  The library contains 50,000 BAC clones with an average insert size of 110 kb.  The Pisum genome is estimated to be 3947 to 4397 Mbp/1C as determined by flow cytometry (3) so we estimate this library represents one haploid genome equivalent.  In addition, we have packaged 20,000 mini-BACs (£60 kbp) which may be useful in filling gaps in a 5- to 6-fold coverage library. The final library of PI 269818 of approximately 250,000 clones will represent five to six-fold haploid genome equivalents, depending on the average insert size of the BAC clones.  Map-based cloning using large-insert libraries has been useful for identifying candidate plant resistance genes (2).  The binary vector, V41, allows for the direct transformation of candidate gene BACs into plants to verify the phenotypic effect of the putative function of the gene.

      The BACs will be sequenced for targeted microsatellite development, recently demonstrated in soybean (4, 9).  Markers linked to traits of interest are used as probes to identified  BACs which will be sequenced to discover microsatellites around the marker loci.  The sequence data will be used to first identify the simple sequence repeats (SSR) and second, design primers around the SSR to convert the information into a co-dominant PCR marker.

      These markers, called sequence-tagged microsatellites (STMS) will be useful for genetic mapping, marker-assisted selection for genes underlying resistance, and fingerprinting and assessing genetic diversity in pea germplasm resources.  The systematic sharing of the BAC sequence information and published microsatellites markers will potentially speed the discovery of positive allele/loci available in the Western Regional Plant Introduction Station Pisum sativum germplasm collection.  DNA has been isolated from most of the accessions in the NPGS and is available for STMS analyses.  Now that the  50,000 large insert pea BAC clones are packaged,  mapped pea probes will be used to identify BACs containing the markers to verify that the library is representative of the genome (6).  The library will be available from the corresponding author (coynec@wsu.edu) for collaborative research with pea geneticists.

Acknowledgment: The authors would like to gratefully acknowledge the technical assistance of Jeff Shultz, Leon Razai and Eowyn Corcoran Elan.  This research was funded in part by grants from the Northwest Agriculture Research Foundation to Coyne and Inglis, USDA-CREES Special Grant program for Cool Season Food Legumes to Inglis, Coyne and McPhee and contribution form Southern Illinois University at Carbondale (Meksem and Lightfoot).

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